DESCRIPTION (Applicant's Description): Cytosolic Ca2+, the primary mediator of smooth muscle cell contraction, is controlled through a combination of Ca2+ release from endoplasmic reticulum (ER) Ca2+ stores and entry of Ca2+ across the plasma membrane (PM). Although there are differences in the relative roles of these two Ca2+ sources among different smooth muscle subtypes, intracellular Ca2+ stores play a crucial role in controlling the generation of Ca2+ signals in response to activation of phospholipase C-coupled receptors. Despite their importance, Ca2+ stores remain poorly defined and elusive entities within smooth muscle cells as well as most nonmuscle cell types. The objective is to study the function and organization of Ca2+ stores and their intricate relationship with the PM in the generation of Ca2+ signals. A central hypothesis to be evaluated is that direct coupling between Ca2 about stores and the PM is required in the activation of Ca2+ entry signals. The studies utilize a combination of established smooth muscle cell lines, primary cultures of aortic smooth muscle cells, and transfected model cell systems to assess the following questions: 1. What is the Nature of the Coupling Between Ca2+ Stores and the Plasma Membrane? Much evidence indicates that direct interactions between Ca2+ stores and PM mediate receptor-activated Ca2+ signaling events. Our objective is to probe the molecular components involved in this interaction. The specific hypothesis to be tested is that the InsP3 receptor is a central component in mediating this interaction. The experimental approach is to assess the role of different subtypes and molecular domains of the InsP3 receptor in mediating the coupling between ER and PM to activate store-operated Ca2+ entry channels. Experiments will investigate the molecular targets of the InsP3 receptor including members of the ubiquitously expressed TRP (transient receptor potential) family of receptor-activated cation entry channels and their role in mediating Ca2+ entry in response to store-depletion. 2. How are Ca2+ Stores Functionally Organized in Smooth Muscle Cells? Studies indicate that Ca2+ stores are organized in an intricate, heterogeneous, and dynamic network within cells. Our aim is to use a combination of biochemical, molecular, and cellular imaging approaches to probe the functional organization of Ca2+ stores. Central within this aim is to test the hypothesis that functionally identifiable coupling occurs between specific Ca2+ stores and discrete areas of the PM to mediate the activation of Ca2+ entry channels. The approach is to investigate the cellular components involved in this coupling, including the exocytotic machinery mediating trafficking and docking, and the role of cytoskeletal elements. Functional studies will be complemented with high-resolution imaging to spatially localize Ca2+ stores and Ca2+ signaling events. The studies represent a cohesive combination of cell biological and molecular approaches to determining how Ca2+ stores function to generate cytosolic Ca2+ signals - key to controlling the contractile responses and proliferation of smooth muscle cells. The mechanisms by which Ca2+ signals are generated is essential to understanding and modifying major vascular diseases involving functional aberrations or changes in the proliferation of vascular smooth muscle cells.